Interferon (hereinafter, the “interferon” may be abbreviated as IFN) is the most important cytokine in the anti-virus immune response. Interferon producing cell in human blood (IPC: IPC is an undifferentiated lymphocyte-based dendritic cell positioned as a precursor cell of the dendritic cell (DC). The IPC may be also called plasmacytoid dendritic cell or plasmacytoid dendritic cell (pDC). Hereinafter, in the present specification, IPC and pDC are synonymous, and uniformly referred to as a term of pDC in principle below.) expresses CD4 and major histocompatible complex class II protein. However, isolation or particular characterization of the cells has not been performed until now due to an insufficient number of such cells, rapid apoptosis, and further lack of lineage (system) marker. It has been revealed that pDC is CD4+CD11c−2 type precursor cell of the dendritic cell, and found out that pDC produces IFN more by 200 to 1000 folds than other blood cells after stimulation by a microorganism. Accordingly, pDC is a conclusive immune system effector cell in an anti-virus/anti-tumor immune response.
IFNα and IFNβ are known as type I IFN having anti-virus activity or anti-tumor activity. On the other hand, it has been revealed that IFNα is associated with autoimmune diseases. For example, abnormal production of IFNα has been reported in patients of autoimmune diseases such as systemic lupus erythematosus and chronic rheumatoid arthritis. Furthermore, it has been reported a case where autoimmune disease symptoms are expressed or aggravated upon administration of a recombinant IFNα2 or IFN. It has been also suggested that autoimmune symptoms are likely to be alleviated by neutralization of IFNα.
In addition, it has been also revealed that IFNα induces differentiation of dendritic cell (DC). It has been contemplated that induction of differentiation of a dendritic cell constitutes an important mechanism in an autoimmune disease since a dendritic cell is an antigen presenting cell. In fact, it has been suggested that induction of differentiation of a dendritic cell of IFNα is deeply associated with development of systemic lupus erythematosus. As described above, close relationship of IFNα with autoimmune diseases as well as anti-tumor activity has been pointed out. In addition, IFNα is also deeply associated with development of psoriasis.
Only a few pDC exists in the blood. It is contemplated that the ratio of pDC occupying the peripheral blood lymphocyte is 1% or less. However, pDC has very high IFN-production ability. The IFN-production ability of pDC reaches, for example, 3000 pg/mL/104 cells. That is to say, it can be said that most of IFNα or IFNβ in the blood produced at the time of virus infection is caused by pDC, although the number of the cells is small.
pDC is differentiated into a dendritic cell by virus stimulation, and induces production of IFN-γ or interleukin (IL)-10 by T cell. In addition, pDC is also differentiated into a dendritic cell by IL-3 stimulation. The dendritic cell differentiated upon IL-3 stimulation induces production of Th2 cytokine (IL-4, IL-5, IL-10) by T cell. As described above, pDC has a property that it is differentiated into different dendritic cells depending on the difference of stimulations.
Accordingly, pDC is a cell that has two sides, i.e., one side as an IFN producing cell, and the other side as a precursor cell of a dendritic cell. Either one of the cells plays an important role in the immune system. That is to say, pDC is one of the important cells that support the immune system in various aspects.
In regulation of the activity of a humoral factor such as IFN, administration of an antibody that recognizes the factor is effective. For example, an attempt to treat autoimmune diseases with an antibody against IL-1 or IL-4 was in practical use. In addition, also for IFN similarly, neutralization antibody is regarded as a therapeutic agent for autoimmune diseases. It can be expected that similar approach is effective for IFN producing pDC. However, such approach is based on inhibition of the action of a humoral factor after being produced. If production of an intended humoral factor can be directly controlled, further essential therapeutic effects can be achieved.
Antibodies that recognize human pDC have been reported. For example, anti-BDCA-2 monoclonal antibody is a human pDC-specific monoclonal antibody (Dzionek, A. et al. J. Immunol, 165: 6037-6046, 2000). It has been revealed that the anti-BDCA-2 monoclonal antibody has an action of suppressing IFN production of human pDC (J. Exp. Med. 194: 1823-1834, 2001). Additionally, it has been also reported that a monoclonal antibody that recognizes mouse interferon-producing cell suppresses the production of interferon (Blood 2004 Jun. 1; 103/11: 4201-4206. Epub 2003 December). It has been also reported that the number of dendritic cells decreases by a monoclonal antibody for mouse pDC (J. Immunol. 2003, 171: 6466-6477).
Similarly, it would be useful if an antibody that can recognize human pDC and regulate the activity thereof is provided. For example, the present inventors revealed already that an antibody recognizing Ly49Q specifically binds to mouse pDC. However, the antibody for Ly49Q did not interfere with the activity of mouse pDC (Blood, 1 Apr. 2005, Vol. 105, No. 7, pp. 2787-2792).
PLD is an enzyme that catalyzes a reaction of hydrolysis of phosphatidyl choline to produce phosphatidic acid and choline, and causes signaling in various cells. It is contemplated that the produced phosphatidic acid functions as a lipid signal molecule.
PLD1 and PLD2 are conventionally known as two kinds of mammal PLDs, and contain Phox homology domain (PX domain), which is bondable to phosphatidyl inositide, and pleckstrin homology domain (PH domain) at the N terminal region thereof. Both of the domains are involved in PLD membrane targeting.
PLD1 and PLD2 further contain two His-x-Lys-x-x-x-x-Asp sequences (HKD motif). This HKD motif is an essential domain in PLD activity.
It has been contemplated that phosphatidic acid produced by PLD1 and PLD2 is involved in re-constitution of cellular skeleton, exocytosis, phagocytosis, canceration, cell adhesion, chemotaxis, and the like, and acts centrally in the nerve system, the immune system, and the like.
Although human Hu-K4 and mouse SAM9 are officially named as PLD3 until now, they are lack of PX and PH domains, and exhibit no PLD activity though they have two HKD motifs. Furthermore, although there are three PLD family members, i.e., PLD4, PLD5, and PLD6, these nonclassical PLDs are scarcely known.
The cerebellar development transcriptome database (CDT-DB) for gene expression pattern in development of mouse cerebellum was searched, and as a result thereof, PLD4, which was a transcription product that was controlled at the time of development, was identified (see Tao et al., Nat. Methods 2(8), 591-598 (2005)). Basic characteristics of PLD4 have not been reported. It is regarded that it should be determined from now whether PLD4 exhibits enzymatic activity or not, and whether a de-glycosylated form of PLD4 has PLD activity or not.
PLD4 is a 506 amino acid sequence represented by SEQ ID NO: 1 (Tao et al., Nat. Methods 2(8), 591-598(2005) and Clark et al., Genome Res. 13(10), 2265-2270(2003)). The PLD4 protein has two tentative PDE regions (phosphodiesterase motif), which are constituted with two HKD motifs (amino acid sequence of His-x-Lys-x-x-x-x-Asp, wherein x is the other amino acids) conserved in the C terminal region, and a presumptive phosphorylation site (Thr 472). The structure of the PLD4 protein is predicted as a type II monotropic transmembrane protein. In addition, the N terminal region of the PLD4 protein does not have PX region and PH region, which are possessed by PLD1 and PLD2 that are classical PLD family (FIGS. 1 and 2).
On the other hand, although PLD4 belongs to the PLD family from the fact that it has two HKD motifs, PLD4 is lack of PX domain and PH domain, but has a putative transmembrane domain instead.
mRNA expression of PLD4, which was characteristically at from a low level to a medium level, was found in a cell subpopulation that was preferentially localized at the corpus callosum and the periphery of the white matter region including cerebellar white matter of a mouse 1 week after birth. These cells expressing the PLD4 mRNA have been identified as Ibal positive microglia (see Tao et al., Nat. Methods 2(8), 591-598 (2005)).
The period of 1 week after birth is a period when activation of myelin formation starts in the corpus callosum and the cerebellar white matter of a mouse. In this period, PLD4 is highly expressed at the amoeboid (activated state) microglia that exists in the white matter. From these facts, a possibility is contemplated that PLD4 expression cell in the white matter is involved in myelin formation in this period. Particularly, accumulation PLD4 in the phagocytic vesicle becomes evident, and a possibility is suggested that PLD4 expression cell is involved in phagocytosis. From the amoeboid microglia in the activated state, various cytokines or growth factors are secreted, and phagocytosis is activated as well. It is contemplated that extra oligodendrocyte (glia cell in the central nervous system, which forms myelin as rolled and attached to the axon) causes apoptosis in the white matter of the brain at the development stage. A possibility has been contemplated that the extra oligodendrocyte is degraded and removed from the amoeboid microglia to secret a signal molecule, whereby to arrange the environment for myelin formation in the white matter. It is suggested that PLD4 is involved in these processes including the myelin formation.
PLD4 mRNA expression is universally seen also in non-nerve tissues, but is mainly distributed in the spleen. Strong PLD4 expression is detected at the periphery of the border zone of red pulp of the spleen, and splenic PLD4 protein collected from the membrane fraction in the cell is highly N-glycosylated. When PLD4 was expressed in a heterogeneous cell system, they were localized in the endoplasmic reticulum and the Golgi body. Heterologously-expressed PLD4 showed no PLD enzymatic activity (Plos ONE www.plosone.org, November 2010, Volume 5, Issue 11, e13932).
From the pattern of the PLD4 expression limited in terms of time and location, it is suggested that PLD4 plays a role in common functions in the microglia or the cell in the spleen border region at the time of brain development at the initial stage after birth.
The PLD4 mRNA expression and PLD4 distribution in the nerve tissue and non-nerve tissue have been overviewed above. However, the present inventors found out that PLD4 mRNA is specifically highly expressed in a pDC cell at the resting stage (resting pDC) in the level of a cell species described below.
Mouse anti-human PLD4 polyclonal antibody against total length human PLD4 protein is commercially available (PLD4 purified MaxPab mouse polyclonal antibody (B01P), catalog No. H00122618-B01P, manufactured by Abnova Corporation). However, a monoclonal antibody that binds only to a certain site of PLD4, or a monoclonal antibody that can specifically bind to PLD4, has not been obtained.